PROJECT SUMMARY Type 2 inflammation occurs during intestinal helminth parasite infection and allergic disease, which together affect billions of people worldwide and cause significant morbidity. Hookworm-like parasites migrate through the lung and then reside in the gut, providing a valuable model to study Type 2 inflammation in multiple tissues. The current paradigm suggests that Type 2 inflammation is characterized by epithelial cell production of interleukin (IL)-33 that activates immune cells to produce IL-4 and IL-13, which act back on epithelial cells to promote mucin secretion and worm expulsion. In parallel, synthesis of the bioactive lipid prostaglandin D2 (PGD2) is increased and expression of its receptor CRTH2 is also dynamically regulated on immune and epithelial cells. However, our current cytokine-centric paradigm does not adequately account for how PGD2 and CRTH2 control cell- and tissue-specific responses in Type 2 inflammation. Our preliminary studies leverage a novel CRTH2 reporter mouse that also allows for cell lineage-restricted CRTH2 deletion, revealing CRTH2 expression in group 2 innate lymphoid cells (ILC2s) and intestinal epithelial cells (IECs). We show that ILC2s, critical IL-13-producers, failed to accumulate in the lung during Type 2 inflammation in CRTH2-deficient mice, likely due to effects on ILC2 migration. In contrast, PGD2 counteracted the effects of IL-13 on CRTH2- expressing IECs during intestinal helminth infection. Intestinal tuft cells, epithelial cells that produce anti- helminth IL-25 and leukotrienes, were particularly enriched for CRTH2 expression, and surprisingly, were capable of PGD2 production. These data led us to hypothesize that cell type- and tissue-specific signals control CRTH2 expression in the lung vs. intestine to dictate the context-dependent effects of PGD2 during Type 2 inflammation. To test this hypothesis, we propose 2 Specific Aims that will employ murine models of hookworm-like parasite infection and in vitro murine and human small intestine enteroid cultures. Aim 1 will dissect the role of IL-33 in upregulating ILC2 CRTH2 expression and mechanisms by which the PGD2-CRTH2 pathway controls ILC2 tissue distribution and activation in pulmonary Type 2 inflammation. Aim 2 will test if IL- 13 promotes tuft cell CRTH2 upregulation, how the PGD2-CRTH2 pathway regulates pro-inflammatory cytokine and leukotriene release from murine and human tuft cells, how tuft cell-intrinsic CRTH2 regulates protection against helminth infection, and if tuft cells are a critical source of PGD2 in the intestine. These studies will reveal how PGD2 and CRTH2 control Type 2 inflammation. Trials employing CRTH2 inhibitors to treat allergic respiratory disease in humans have yielded conflicting results, potentially attributable to unappreciated effects of CRTH2 on a variety of immune and epithelial cell types. Thus, our studies stand to inform the strategic use of CRTH2 inhibitors in the modulation of lung and inte...